The long-term objective of this research is to understand the mechanism by which intermixing of the human long- (L) and middle- (M) wavelength cone photopigment genes give rise to variants that cause aberrant pre- messenger RNA (mRNA) splicing, and lead to vision loss with a diverse set of clinical phenotypes. 95% of our cones are L or M cones, and except at very low light levels when rods are active, all vision is based on cones. The L and M cones play a critical role in the visually guided feedback mechanism responsible for controlling eye growth (emmetropization). Every aspect of seeing, including high acuity and color vision, depends on the L and M cone photopigments and these genes are important risk factors in common eye disorders that plague modern humans. The L and M (LM) cone photopigment genes, designated OPN1LW and OPN1MW, respectively, exhibit high haplotype diversity in exons 2, 3 and 4. The most is known about two variants, designated LIAVA and LVAVA that are associated with photoreceptor dysfunction and severe vision impairment. They are found in patients with a range of clinical diagnoses including high grade myopia, blue cone monochromacy and cone dystrophy. We and others have recently shown that combinations of single nucleotide polymorphisms (SNPs) associated with these variants cause aberrant pre-mRNA splicing. Our preliminary data show that different combinations of the exon 3 polymorphisms shift the ratio of full length to exon 3 skipped mRNA, producing variability in the severity of the splicing defect that will be extremely useful in elucidating the fundamental mechanisms controlling splicing of this exon. Preliminary data obtained using a cell culture-based splicing assay also suggests that haplotypes of exon 3 that yield moderate levels of exon 3 skipping are associated with an average -1.3 diopters of refractive error compared to low/non-skipping variants. The pathophysiology of the different mutations is complex because superimposed on the effects of subnormal amounts of opsin protein produced by the splicing defects, are the effects of some combinations of the amino acids on protein function. To achieve our goal we propose: Aim 1: To investigate the role of combinations of SNPs in exons 2, 3 and 4 of the L and M opsin genes in splicing by 1.1 fully enumerating the transcript isoforms, and measuring their relative abundances. 1.2 investigating and quantitating the effects of exon 3 haplotypes on splicing in cone photoreceptors and particularly those haplotypes that are risk alleles for juvenile onset myopia. Aim 2: To investigate the mechanism of exon 3 skipping using biochemical and molecular biology approaches Aim 3: To evaluate the potential for exon specific U1 snRNAs to rescue the exon 3 skipping phenotype.